Domain organization of D-AKAP2 revealed by enhanced deuterium exchange-mass spectrometry (DXMS)
Domain organization of D-AKAP2 revealed by enhanced deuterium exchange-mass spectrometry (DXMS)
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DOI：
10.1016/s0022-2836(02)00419-9
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发表时间：
2002-08-23
影响因子：
5.6
通讯作者：
Woods, VL
中科院分区：
文献类型：
--
作者：
Hamuro, Y;Burns, LL;Woods, VL
双特异性A-激酶锚定蛋白2（D-AKAP 2）是通过结合I型和II型蛋白激酶A（PKA）来协调cAMP介导的信号传导复合物的支架蛋白。虽然有关特异性结合基序的信息正在展开，但对这些支架蛋白的整体结构和动力学知之甚少。我们已经使用氘交换质谱（DXMS）和有限的蛋白水解来探测D-AKAP 2的折叠区域，首次提供了对支架蛋白的结构域内动力学的洞察。氘交换揭示了两个低氘交换区域，其被高交换区域包围，这表明两个明显折叠的区域，两侧是无序或溶剂可访问的区域。通过有限的蛋白水解检测到类似的折叠区域。第一个折叠区含有一个假定的G蛋白信号调节子（RGS）结构域。RGS结构域的结构模型显示，更多的氘代区域映射到环和转角上，而较少的氘代区域映射到α-螺旋上，这与该区域折叠成RGS结构域一致。第二个折叠区域包含一个高度保护的PKA结合位点和一个更易被溶剂接近的PDZ结合基序，这可能是D-AKAP 2的潜在靶向结构域。DXMS验证了序列同源性暗示的D-AKAP 2的多结构域结构，并提供了对PKA结合位点可及性的独特见解。(C)2002爱思唯尔科技有限公司版权所有。
Dual specific A-kinase anchoring protein 2 (D-AKAP2) is a scaffold protein that coordinates cAMP-mediated signaling complexes by binding to type I and type II protein kinase A (PKA). While information is unfolding regarding specific binding motifs, very little is known about the overall structure and dynamics of these scaffold proteins. We have used deuterium exchange-mass spectrometry (DXMS) and limited proteolysis to probe the folded regions Of D-AKAP2, providing for the first time insight into the intra-domain dynamics of a scaffold protein. Deuterium on-exchange revealed two regions of low deuterium exchange that were surrounded by regions of high exchange, suggestive of two distinctly folded regions, flanked by disordered or solvent accessible regions. Similar folded regions were detected by limited proteolysis. The first folded region contained a putative regulator of G-protein signaling (RGS) domain. A structural model of the RGS domain revealed that the more deuterated regions mapped onto loops and turns, whereas less deuterated regions mapped onto alpha-helices, consistent with this region folding into an RGS domain. The second folded region contained a highly protected PKA binding site and a more solvent-accessible PDZ binding motif, which may serve as a potential targeting domain for D-AKAP2. DXMS has verified the multi-domain architecture Of D-AKAP2 implied by sequence homology and has provided unique insight into the accessibility of the PKA binding site. (C) 2002 Elsevier Science Ltd. All rights reserved.